IntAct: A nondisruptive internal tagging strategy to study the organization and function of actin isoforms

Author:

van Zwam Maxime C.,Dhar Anubhav,Bosman Willem,van Straaten Wendy,Weijers Suzanne,Seta Emiel,Joosten Ben,van Haren Jeffrey,Palani Saravanan,van den Dries KoenORCID

Abstract

Mammals have 6 highly conserved actin isoforms with nonredundant biological functions. The molecular basis of isoform specificity, however, remains elusive due to a lack of tools. Here, we describe the development of IntAct, an internal tagging strategy to study actin isoforms in fixed and living cells. We identified a residue pair in β-actin that permits tag integration and used knock-in cell lines to demonstrate that IntAct β-actin expression and filament incorporation is indistinguishable from wild type. Furthermore, IntAct β-actin remains associated with common actin-binding proteins (ABPs) and can be targeted in living cells. We demonstrate the usability of IntAct for actin isoform investigations by showing that actin isoform-specific distribution is maintained in human cells. Lastly, we observed a variant-dependent incorporation of tagged actin variants into yeast actin patches, cables, and cytokinetic rings demonstrating cross species applicability. Together, our data indicate that IntAct is a versatile tool to study actin isoform localization, dynamics, and molecular interactions.

Funder

Department of Biotechnology-Wellcome Trust India Alliance

Science and Engineering Research Board

Indian Institute of Science

Erasmus Medisch Centrum

Nederlandse Organisatie voor Wetenschappelijk Onderzoek

Publisher

Public Library of Science (PLoS)

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